Fig 2: ERAP1 inhibition impairs Hh-dependent tumor growth in vivo. a–f NSG mice were grafted with spontaneous primary MB from Math1-cre/PtcC/C mice. Tumor masses (150 mm3) were intratumorally injected with Leu-SH. a Tumor growth was monitored. b Representative flank allograft average volumes (lower panel) and quantification of tumor explants (upper panel). c, d Ki67, NeuN, and cleaved Caspase-3 (Cl.Casp-3) immunohistochemical stainings of allograft tumor samples. d Quantification of immunohistochemical stainings shown in c. Scale bar 100 µm. e mRNA and f protein expression levels of Hh targets from tumors assayed in b. g Representative H&E images (low and high magnification) of a murine MB cell-derived orthotopic tumor in NSG mice after i.p. injection of Leu-SH. Scale bars, 500 µm and 200 µm (upper and lower panels, respectively). h Representative average volume of orthotopic tumor. i–j NSG mice were grafted with spontaneous primary MB from Math1-cre/PtcC/C mice genetically silenced for ERAP1 expression. i Representative images of mice and the explanted tumor masses. j Quantification of the flank allograft average tumor volume. ERAP1 protein expression is shown below. In f, j, actin was used as loading control. k H&E and representative Masson’s trichrome staining of tumors. Scale bar 100 µm. l mRNA levels of the indicated Hh target genes. m Representative H&E images (low and high magnification) of a murine MB cell-derived orthotopic tumor genetically interfered for ERAP1 before the injection in NSG mice cerebella. Scale bars, 500 and 200 µm (upper and lower panels, respectively). n–p ERAP1 accelerates Hh-MB formation. n Tumor volume of mice subcutaneously transplanted with GCPs from tumor-prone Math1-cre/PtcC/C animals overexpressing ERAP1. o Representative flank allograft average volumes (lower panel) and quantification of the explanted tumor masses (upper panel). p mRNA expression of Hh target genes from the tumor masses assayed in o. q Survival curves of Math-cre/PtcC/C mice treated with Leu-SH or vehicle. Results in e, l, p were normalized to endogenous GAPDH and HPRT controls and expressed as in Fig. 1. All data represent the mean of three independent experiments. Mean ± S.D. of tumor (n = 6) for each treatment. *P < 0.05, **P < 0.01, *** P < 0.001 calculated by two-sided Student’s t-test

Fig 3: Defective Shh signaling induced by axoneme hypoglutamylation can be restored by concomitant CCP5 depletion. a hTERT-RPE-1 cells were treated with the indicated siRNAs for 48 h, serum starved, and treated with 500 nM SAG for 24 h. GLI3 ciliary localizations were examined by immunofluorescent staining. Percentages of the cilia with GLI3 at cilia tip were quantified. Scale bar: 2 µm. b Depletion of either FIP5 or TTLL6 abolished GLI1 expression upon hedgehog activation. hTERT-RPE-1 cells were treated with the indicated siRNAs for 48 h, serum starved, and treated with 500 nM SAG for 24 h. The relative mRNA levels of GLI1 were measured by real-time PCR. c Depletion of CCP5 suppressed GLI3 localization to cilia tip. Percentages of the cilia with GLI3 in cilia tip were quantified. d Depletion of CCP5 restored the ciliary tip localization of GLI3 in FIP5 or TTLL5-depleted hTERT-RPE-1 cells. Percentages of the cilia with GLI3 in cilia tip were quantified. e Depletion of CCP5 rescued GLI1 expression induced by SAG as seen by real-time PCR. Data shown in a–e are the statistical analysis of three independent experiments. f Schematic model of polyglutamylation regulation and function in the context of cilia. During the early stage of ciliogenesis, ARL13B recruits RAB11/FIP5-positive vesicles, and then RAB11/FIP5-positive vesicles tether TTLL5/6-containing vesicles to the proximity of the ciliary base, which allowing cilia import of TTLL5/6. Inside cilia, axoneme polyglutamylation was controlled by the balanced enzymatic activities between TTLLs (mainly TTLL5 and TTLL6) and ciliary deglutamylase CCPs (CCP5 and other CCPs that shorten the long glutamate chains). Polyglutamylated axoneme anchors sensory receptor polycystins to the ciliary surface via unknown adaptor mechanism, which could interact with both the glutamylated chain and the intercellular domain of Polycystins. Additionally, axoneme polyglutamylation is required for proper translocation of hedgehog signaling molecule GLI3 to the ciliary tip, likely through a different adaptor mechanism that can recognize both polyglutamylated chain and intraflagellar transport machinery. Center values represent mean. Error bars represent s.d. Statistical significance was determined using two-way ANOVA test. * p < 0.05; ** p < 0.01. n.s. not significant

Fig 4: TALPID3 and ANKRD26 orchestrate the cilia gating for membrane receptors.The localizations of PKD2 and Shh signaling pathway-associated proteins were analyzed in control, TALPID3 single-knockdown, ANKRD26 single-knockdown and TALPID3 and ANKRD26 double-knockdown RPE cells. Cells were examined by IF staining after 24 h of serum starvation with the indicated antibodies. a Compared with WT, TALPID3 single-knockdown, and ANKRD26 single-knockdown cells, TALPID3 and ANKRD26 double-knockdown cells showed significantly reduced ciliary entry of PKD2. Right panel, both the percentages of cells with PKD2 ciliary signaling and the mean fluorescence intensity in each group were quantified. For PKD2-positive ratio, n = 300 cells over three independent experiments. For PKD2 intensity, numbers of cell examined are indicated above each dataset. b Cells treated without (-SAG) or with (+SAG) SAG were compared to analyze Gli3 and Smoothened (Smo) localization. c The percentages of cells with ciliary Gli3 and Smo were quantified. Results from a minimum of 100 cells in each group were averaged. The results from three independent experiments were statistically analyzed and plotted. n = 300 cells over three independent experiments. d A prospective model of the regulation of FBF1 by TALPID3 and ANKRD26. Recruitment of FBF1 to DAs/TFs or its stabilization on DAs/TFs requires coordination between the distal centriole wall and DA blades. Deletion of TALPID3 compromises the condition of the distal centriole wall, and deletion of ANKRD26 may affect the condition of the DA blade; therefore, co-depletion of TALPID3 and ANKRD26 exacerbates defects in FBF1 localization. All data are presented as the mean ± s.d. Significant differences were identified by two-tailed unpaired Student’s t test. NS, P > 0.05; ***, P < 0.001. No adjustments were made for multiple comparisons. Scale bars, 2 µm. Source data are provided as a Source Data file.

Fig 5: Model of compartment specific functions of Tbx3 in forelimb bud mesenchyme and altered interactions and stoichiometry of the Kif7/Sufu Gli3 processing complex in Tbx3;PrxCre mutants.In posterior forelimb mesenchyme, Tbx3 is required for normal levels of Hand2 upstream of Shh. Shh pathway activity and other Tbx3-reponsive factors promote digit 5 formation. In the absence of Tbx3, there is decreased expression of Hand2 and Shh and other digit 5 promoting pathways. In anterior mesenchyme, Tbx3 is in a complex with Gli3 proteins, Kif7 and Sufu and required for the stability of Gli3 FL and Gli3R. In the absence of Tbx3, Sufu and Spop protein levels are increased yet there is decreased interaction between Sufu and Kif7, and Sufu and Gli3. In mutant anterior mesenchyme, Gli3FL is barely detected: it is either degraded or converted to Gli3R. Levels of Gli3R are abnormally low due to a combination of decreased amount of Gli3FL precursor and its processing to Gli3R, and excess degradation. These findings are consistent with decreased function of cilia and Kif7 (required for processing of Gli3FL to Gli3R), and of Sufu (required for stability of both Gli3FL and Gli3R).DOI: http://dx.doi.org/10.7554/eLife.07897.042